Summary Helminth parasites remain one of the world?s greatest challenges to human and animal health, with more than two billion infected humans world-wide. Parasite control is restricted to short-term suppression with low cost drugs due the unavailability of effective vaccines. Persistence of helminths in humans and animals is testament to their highly evolved ability to evade the immune system, thus understanding the evasion mechanisms is key to developing new interventions. In this proposal, the focus is on the mouse parasite Heligmosomoides polygyrus, which infects animals as L3 larvae and colonizes and matures in the intestine. In mice infected with H. polygyrus, there is a dramatic upregulation in the number of Foxp3+ regulatory T-cells (Tregs), which broadly and potently mediate immune suppression. Interference of Treg function in infected mice results in expulsion of H. polygyrus, demonstrating that the expanded pool of Foxp3+ Tregs is essential for persistence. In collaboration with the Maizels group, we have shown that a secreted five-domain complement control protein (CCP) protein that mimics mammalian TGF-b, a cytokine that potently suppresses the immune system by stimulating the proliferation and differentiation of Tregs, is critical for immune hyporesponsiveness in H. polygyrus infected animals. In spite of lacking any homology to mammalian TGF-?, this protein termed TGF-b mimic or TGM, binds directly to the mammalian TGF-? receptors, T?RI and T?RII, to induce signaling and downstream effects on Tregs. In this proposal, we will determine how TGM binds and assembles T?RI and T?RII into a signaling complex, with the goal of using this information to better understand how TGM signals and identify homologous proteins in other parasites that exploit the TGF-b pathway to evade host immune responses. In addition, this information can be leveraged to engineer forms of TGM for: 1) treating autoimmune diseases, such as rheumatoid arthritis, multiple sclerosis, or asthma, 2) suppressing the immune system in organ transplantation, and 3) developing anti-parasitics that function by blocking the interaction between TGM and the TGF-? receptors. In support of the proposed studies, we show using NMR and ITC/SPR binding studies that TGM domain 3 (TGM-D3) is the primary domain responsible for binding TbRII, while TGM domain D2 (TGM-D2), together with a more minor contribution from TGM domain 1 (TGM-D1), is responsible for binding TbRI. In order to accomplish the objectives of the proposed research, we will determine the structures of TGM-D2 and TGM-D3 alone, and the TGM-D2:TbRI and TGM-D3:TbRII complex structures, using NMR in Aim 1, and the structure of the TGM-D123:TbRII:TbRI complex using X-ray crystallography in Aim 2. In order to identify the residues that contribute greatest to receptor binding, site-directed mutagenesis and ITC- and SPR-based binding studies will be used, together with functional studies in cultured TGF-b reporter cell lines and Tregs.